H6亚型禽流感病毒一步法RT-PCR检测方法的建立

Development of one step RT-PCR technique for detection of H6 subtype avian influenza virus

为快速检测临床样品中的H6亚型禽流感病毒(AIV),本研究通过分析流感数据库中H6亚型的HA基因,在HA保守区设计一对引物,建立一种用于扩增H6亚型禽流感病毒HA基因的一步法RT-PCR检测方法,扩增片段为327 bp。采用该方法对H6亚型AIV的尿囊液10倍倍比稀释样品进行检测,结果显示最低检出量为102.5EID50/mL。用该方法检测其他亚型AIV和鸡新城疫病毒等病原均为阴性,具有良好的特异性。对H6 AIV人工感染鸡的咽喉、泄殖腔棉拭子样品进行一步法RT-PCR检测,并与病毒分离进行比较,显示该方法对棉拭子的检测极限可达102.5EID50/mL,同时利用该方法及病毒分离对临床样品进行检测,两者检测结果一致。实验结果表明该RT-PCR方法具有较好的特异性、敏感性,可以应用于临床样品的实验室检测。

To establish a rapid detection method for H6 subtype of avian influenza virus （AIV）, one step RT-PCR assay was developed to amplify the AIV HA gene with a pair of specific primers designed based on H6 subtype AIV. The amplification product of the assay was 327 bp in size and the detection limit was 102.5 EID^mL in allantoic fluid of H6 subtype AIV infected embryo eggs by this assay, but no amplification for other subtypes of AIV. In addition, Swab samples from H6 subtype AIV experimental infected chicks were tested and the results by the one step RT-PCR detections were completely consistent with the conventional virus isolation method. Therefore, the one step RT-PCR method established in this study is highly specific and sensitive for the detection of H6 subtype AIV.